灰飞虱LHSC70基因表达特性及功能

doi:10．3969/j．issn．1001G7216．2015．04．012

摘要/Abstract

摘要：

灰飞虱是我国主要水稻害虫之一,在各水稻种植区均有分布,对环境具有较强的适应性。HSP70作为分子伴侣在生物生长发育和外界刺激应激中发挥重要作用。HSC70是HSP70家族重要成员之一,具有组成型表达特性。为探讨灰飞虱对环境适应的分子机理,本研究采用qPCR、RNA干涉等技术研究了灰飞虱HSC70的表达特性和功能。结果表明,灰飞虱HSC70 基因长1971 bp,命名为LHSC70,编码656个氨基酸,推测分子量为71.6 kD,等电点为5.46,含HSP70 家族3个标签序列,具有HSP70典型结构特征。系统进化分析表明LHSC70与褐飞虱HSC70同源性最高,达到99%。LHSC70基因的表达与龄期有关,1~5龄若虫的表达量无显著差异,但雌成虫的表达量明显高于若虫和雄成虫;10、16、22、26、30、34、38、42℃下,雌成虫LHSC70基因表达量变化不显著。RNA干涉抑制雌成虫LHSC70 基因表达,导致其耐热性下降,而产卵量变化不大。这些结果说明灰飞虱LHSC70为组成型热激蛋白70,与组成抗性有关,与繁殖关系不大,且不受热激诱导。

关键词: 灰飞虱, HSC70, 表达量, RNAi, 组成抗性

Abstract:

Laodelphax striatellus is an major pest on rice in China. It has strong adaptability to environments and was found in all rice growing areas of the country. As a molecular chaperone, HSP70 plays an important role in biological growth and response to stress. HSC70,characterized by constitutive expression, is one of the most important members of the HSP70 family. To explore the adaptation mechanisms of L. striatellus to environments, LHSC70 expression profiling and function were analyzed. The results showed the complete cDNA of LHSC70 is 1971 bp in length, which encodes a protein of 656 amino acids, with a molecular weight of 71.6 kD and theoretical isoelectric point of 5.46. LHSC70 contains three signature sequences of HSP70 family and carries a typical HSP70 family structural signature. The phylogenic tree showed it shares 99% homology with HSC70 of Nilaparvata lugens. The expression of LHSC70 was associated with larval instar. The expression level of LHSC70 was highest in female adults and was similar in male adults and larvae at various instars. The difference in the expression of the gene was not significant after 10, 16, 22, 26, 30, 34, 38 and 42℃ treatments. Downregulated expression level of LHSC70 due to RNAi significantly decreased the tolerance of the female to heat stress, while exerted a less influence on the oviposition. These results suggested that LHSC70 is a 70 kD heat shock cognate protein and is not induced by heat stress, but it contributes the constitutive resistance to heat without close relation to reproduction in L. striatellus.

Key words: Laodelphax striatellus, HSC70, expression, RNAi, constitutive resistance

中图分类号:

王利华, 单丹, 方继朝. 灰飞虱LHSC70基因表达特性及功能[J]. 中国水稻科学, 2015, 29(4): 424-430.

Li-hua WANG, Dan SHAN, Ji-chao FANG. Expression Profiling and Function Analyses of LHSC70 in Laodelphax striatellus[J]. Chinese Journal OF Rice Science, 2015, 29(4): 424-430.

图/表 5

表1 qPCR引物碱基序列

Table 1 Primers sequences for qPCR.

基因名 Gene name	引物 Primers	碱基序列(5'-3') Base sequences (5'-3')
LHSC70	Lhsp70-1F	5'-ATTGAGCGCATGGTGAATGACG-3'
	Lhsp70-1R	5'-CCTTCTGCTTGTGCTCGAACTCCT-3'
内参基因 Reference gene	ARFUF	5'-TTGGACAGTATCAAGACCCATC-3'
	ARFUR	5'-GCAGCAATGTCATCAATAAGC-3'

图1 灰飞虱LHSC70氨基酸序列（红色表示HSP70家族标签序列。）

Fig. 1. Amino acid sequence of LHSC70.（Red letters refer to the conserved sequences of HSP70 family.）

图2 灰飞虱LHSC70与其他昆虫或螨HSC70系统进化树分析（HSC70来源及基因登录号:二化螟, AB206478;烟草天蛾, AF194819;摇蚊, AF448433;黑腹果蝇Hsc70-1, Hsc70-2, Hsc70-3, Hsc70-4 和Hsc70-5分别为 NM_079339, NM_079615, NM_167306, NM_079632 和 NM_079017; 丽蝇蛹集金小蜂, NM_001172757;蝶蛹金小蜂Hsc70-1和Hsc70-2分别为 EU340838 和FJ798091; 二斑叶螨, DQ310791;褐飞虱, GU723301; 灰飞虱, KP994340;斜纹夜蛾, HM046611;亚洲玉米螟, HQ434763;小菜蛾, AB214973;棉铃虫, FJ432703;甘蓝夜蛾, AB251896; 家蚕, JF836796; 草地贪夜蛾, AY161271。）

Fig. 2. Phylogenetic tree of HSC70 from Laodelphax striatellus and other species based on neighbor-joining method.（Origin of HSC70 and their GenBank accession numbers: Chilo suppressalis,AB206478; Manduca sexta,AF194819; Chironomus tentans, F448433; Drosophila melanogaster Hsc70-1, Hsc70-2, Hsc70-3, Hsc70-4 and Hsc70-5, NM_079339, NM_079615, NM_167306, NM_079632 and NM_079017; Nasonia vitripennis,NM_001172757; Pteromalus puparum, Hsc70-1 and Hsc70-2, EU340838 and FJ798091; Tetranychus urticae ,DQ310791; Nilaparvata lugens,GU723301; L. striatellus,KP994340; Spodoptera litura,HM046611; Ostrinia furnacalis, HQ434763; Plutella xylostella, AB214973; Helicoverpa armigera,FJ432703; Mamestra brassicae,AB251896; Bombyx mori,JF836796; Spodoptera frugiperda,AY161271.）

图3 灰飞虱LHSC70 在不同龄期若虫及成虫中(A)及在不同温度下(B)的相对表达量

Fig. 3. Relative expression level of LHSC70 at different growth stages (A) and at different temperature (B) in L. striatellus.

图4 RNAi灰飞虱LHSC70 表达量(A)、42℃下热激死亡率(B)、雌成虫繁殖力(C)和寿命(D)（*表示差异显著。）

Fig 4. Relative expression level of LHSC70 (A), mortality at 42℃, oviposition(C) and longevity of female (D) in L. striatellus fed on dsRNA.（* indicates significant difference.）

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